Residues contributing to the Na(+)-binding pocket of the SLC24 Na(+)/Ca(2+)-K(+) Exchanger NCKX2.

Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.

Mapping and functional characterization of the murine smoothelin-like 1 promoter.

BACKGROUND: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene. RESULTS: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence. CONCLUSION: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

Histological and molecular diversity and heterogeneity of precancerous lesions associated with inflammatory bowel diseases.

AIMS: Inflammatory bowel disease (IBD)-associated precancerous lesions may be adenomatous or non-adenomatous with various histomorphologies. We aim to validate the newly proposed classification, to explore the neoplastic nature of the non-adenomatous lesions and to elucidate the molecular mechanisms underlying the different histomorphologies. METHODS: 44 background precursor lesions identified in 53 cases of surgically resected IBD-associated colorectal and ileal carcinomas were reviewed for the histomorphological features (classified into adenomatous, mucinous, sessile serrated adenoma (SSA)-like, traditional serrated adenoma-like, differentiated, eosinophilic and serrated not otherwise specified (NOS)) and analysed for a key panel of colonic cancer-related molecular markers. RESULTS: Approximately 60% of the lesions were adenomatous, of which some had mixed serrated, mucinous or eosinophilic changes. The remaining non-adenomatous lesions, including all other types except SSA-like type, mostly showed mixed features and focal adenomatous dysplasia. KRAS mutation and p53 mutant-type expression were found in about half cases across all types, while PIK3CA mutation only in some of adenomatous and eosinophilic lesions and MLH1/PMS2 loss in a subset of adenomatous, mucinous and eosinophilic but not in differentiated and serrated lesions. SAT-B2 or PTEN loss and IMP3 overexpression were seen in a small subset of lesions. No BRAF, NRAS or EGFR gene mutation was detected in any type. Certain molecular-morphological correlations were demonstrated; however, no single or combined molecular alteration(s) was specific to any particular morphological type. CONCLUSIONS: IBD-associated precancerous lesions are heterogeneous both histologically and molecularly. True colitis-associated adenomatous lesions are unlikely conventional adenomas. Non-adenomatous lesions without frank cytologic dysplasia should also be regarded as neoplastic.

Effects of monovalent cations on Ca2+ uptake by skeletal and cardiac muscle sarcoplasmic reticulum.

Ca(2+) transport by the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) is sensitive to monovalent cations. Possible K(+) binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca(2+) transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca(2+) transport correlated in most cases with their direct effects on SERCA. Choline(+), however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca(2+) transport. Of the monovalent cations tested, only Cs(+) significantly affected the Hill coefficient of Ca(2+) transport (n(H)). An increase in n(H) from approximately 2 in K(+) to approximately 3 in Cs(+) was seen in all of the forms of SERCA examined. The effects of Cs(+) on the maximum velocity of Ca(2+) uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K(+) binding sites of the different forms of the protein.

Cellular localization of the K+ -dependent Na+ -Ca2+ exchanger NCKX5 and the role of the cytoplasmic loop in its distribution in pigmented cells.

NCKX5 is a bidirectional K+ -dependent Na+ -Ca2+ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans-Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2-NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.

Functional characterization and molecular cloning of the K+-dependent Na+/Ca2+ exchanger in intact retinal cone photoreceptors.

Light-dependent changes in cytoplasmic free Ca(2+) are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na(+)/Ca(2+) exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca(2+) measured with optical probes. The exchanger in cones is K(+) dependent and operates both in forward and reverse modes. In the reverse mode, the K(+) dependence of the exchanger is described by binding to a single site with K(1/2) about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods.

A functional approach to understanding the role of NCKX5 in Xenopus pigmentation.

NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.

Molecular characterization, functional expression and tissue distribution of a second NCKX Na+/Ca2+ -K+ exchanger from Drosophila.

The Na+/Ca2+ -K+ exchanger (NCKX) utilizes the inward Na+ gradient and the outward K+ gradient to promote Ca2+ extrusion from cells. Here, we have characterized a second NCKX from Drosophila. Based on its chromosomal location (X chromosome) we have named it Ncxk-x. Three splice variants were isolated with three distinct N-terminal sequences. NCKX-X differs from NCKX proteins described so far in other species by lacking an N-terminal signal peptide. Heterologous expression of the respective cDNA's resulted in NCKX-X protein expression and K+ -dependent Na+/Ca2+ exchange activity for two of the three splice variants. Transcript localization of Nckx-x was investigated and compared with that previously described by us for Nckx30C.

Characterization and in silico mapping of a novel murine zinc finger transcription factor.

Transcription factors play important roles in development and homeostasis. We have completed an embryonic stem cell-based neural differentiation screen, which was carried out with a view to isolating early regulators of neurogenesis. Fifty eight of the expressed sequence tags isolated from this screen represent known transcription factors or sequences containing transcription factor motifs. We have determined the full-length sequence of a novel mouse zinc finger-containing gene (ZFEND; also known as Mus musculus zinc finger protein 358 (Zfp358)) that was identified from this screen. ZFEND has 87% nucleotide and 86% amino acid identity to a previously identified human cDNA, FLJ10390, which is moderately similar to zinc finger protein 135. Northern blotting and RPAs demonstrate highest expression of ZFEND during mid-late mouse embryogenesis. Expression is also observed in several adult tissues with highest expression in heart, brain, and liver. Whole-mount in situ hybridization studies reveal apparent ubiquitous expression of ZFEND during mid-gestation stages (embryonic days 11.5, 12.5), while sections of whole-mount embryos reveal much higher expression levels in the neural folds during neural tube closure and at the boundary between the forelimb buds and the body wall. Bioinformatic analysis maps ZFEND to mouse chromosome 8pter, while FLJ10390 resides on 19p13.3-p13.2, a gene-rich region to which a number of disorders have been mapped. More precise mapping indicates that the involvement of FLJ10390 in atherogenic lipoprotein phenotype, familial febrile convulsions 2, and psoriasis susceptibility cannot be ruled out.

Mutated alleles of the rod and cone Na-Ca+K-exchanger genes in patients with retinal diseases.

PURPOSE: To study the possible involvement of the rod (SLC24A1) and cone (SLC24A2) Na-Ca+K exchanger (NCKX) genes in retinal diseases. METHODS: DNA was collected from unrelated patients with retinal disease, mainly from North America. A human genomic library was screened with the cone NCKX cDNA, and hybridizing clones were sequenced to determine the genomic organization of the SLC24A2 gene. The single-strand conformation polymorphism (SSCP) technique and direct sequencing were used to screen the patients' DNA for mutations in SLC24A1 and SLC24A2. The effect of selected missense changes on protein function was tested by measuring potassium-dependent Na-Ca exchange of the mutant proteins expressed in insect cells. RESULTS: Twenty-seven novel sequence changes were found in the rod NCKX gene, 21 of which are unlikely to be pathogenic, because they did not cosegregate with the disease or did not affect conserved regions of the protein. Of the remaining six, two were frameshift mutations found in one patient each. If translated, these alleles would encode nonfunctional proteins. Three of the six possibly pathogenic mutations were missense changes located in conserved regions, and their protein functions were assayed. Only one (Ile992Thr) had a significantly low level of exchanger function, but it was found in two unrelated patients who were heterozygotes with different retinal diseases, and this mutation could not be unequivocally associated with either disease. The last of the six changes is likely to create a new splice acceptor site. The genomic organization of the cone NCKX gene was determined, and it contained 11 exons with a few splice variants. Fifteen novel sequence changes were identified in the cone exchanger gene in patients with a cone dysfunction or degeneration. Only three of these sequence changes, all missense changes found in heterozygous patients, were considered possibly pathogenic. Functional analysis showed only a slight reduction in the activity of the corresponding mutant proteins. CONCLUSIONS: Although variant alleles of the rod and cone NCKX genes were found, none could be definitively associated with a specific retinal disease. The human phenotype associated with mutant exchanger alleles remains unknown.

The Na/Ca-K exchanger gene family.

Ca(2+) extrusion driven by both the inward Na(+) gradient as well as the outward K(+) gradient is essential for visual transduction in retinal rod and cone photoreceptors because it removes Ca(2+) that enters photoreceptors via the cGMP-gated and light-sensitive channels. We have cloned rod and cone Na/Ca-K exchanger (NCKX) cDNAs from several species, and we have cloned NCKX cDNAs from lower organisms that lack vertebrate-type vision. Although in situ NCKX physiology has only been documented for vertebrate photoreceptors, it is now clear that NCKX gene products have a much broader distribution pattern. Here, we review some of the structural and functional features that have emerged from our studies on different members of the NCKX gene family.

Cell line specific modulation of extracellular aß42 by Hsp40.

Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aß(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aß(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aß(42) toxicity. In this study we show that neither extracellular Aß(42) nor Aß(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aß(42), Western analysis of Aß(42) was performed following external Aß(42) application. Five hours after a conditioning heat shock, Aß(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aß(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aß(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aß(42) indicating that Hsp40 modulation of Aß(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aß(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aß(42). Our data reveal a biochemical link between Hsp40 expression and Aß(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis.

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in motor learning and memory.

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways.

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.

Site-directed disulfide mapping of residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.

The Na+/Ca2+-K+ exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na+ and K+ gradients to extrude cytoplasmic Ca2+. NCKX proteins are made up of two clusters of hydrophobic segments, both thought to consist of five putative membrane-spanning alpha-helices, and separated by a large cytoplasmic loop. The two most conserved regions within the NCKX sequence are known as the alpha1 and alpha2 repeats, and are found within the first and second set of transmembrane domains, respectively. The alpha repeats have previously been shown to contain residues critical for transport function. Here we used site-directed disulfide mapping to report that the alpha repeats are found in close proximity in three-dimensional space, bringing together key functional NCKX residues, e.g., the two critical acidic residues, Glu188 and Asp548. Glu188Cys in the alpha1 repeat could form a disulfide cross-link with Asp548Cys in the alpha2 repeat. Surprisingly, cysteine substitutions of Ser185 in the alpha1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha2 repeat (Ser545, Asp548, and Ser552), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp575, a residue critical for the K+ dependence of NCKX, was shown to be in the proximity of Ser185 and Glu188, consistent with its role in enabling K+ to bind to a single Ca2+ and K+ binding pocket.

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger.

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.

Topology of the retinal cone NCKX2 Na/Ca-K exchanger.

The Na/Ca-K exchanger (NCKX) is a polytopic membrane protein that plays a critical role in Ca(2+) homeostasis in retinal rod and cone photoreceptors. The NCKX1 isoform is found in rods, while the NCKX2 isoform is found in cones, in retinal ganglion cells, and in various parts of the brain. The topology of the Na/Ca-K exchanger is thought to consist of two large hydrophilic loops and two sets of transmembrane spanning segments (TMs). The first large hydrophilic loop is located extracellularly at the N-terminus; the other is cytoplasmic and separates the two sets of TMs. The TMs consist of either five and five membrane spanning helices or five and six membrane spanning helices, depending upon the predictive algorithm used. Little specific information is yet available on the orientation of the various membrane spanning helices and the localization of the short loops connecting these helices. In this study, we have determined which of the connecting loops are exposed to the extracellular milieu using two different methods: accessibility of substituted cysteine residues and insertion of N-glycosylation sites. The two methods resulted in a consistent NCKX topology in which the two sets of TMs each contain five membrane spanning helices. Our new model places what was previously membrane spanning helix six in the cytoplasm, which places the C-terminus on the extracellular surface. Surprisingly, this NCKX topology model is different from the current NCX topology model with respect to the C-terminal three membrane helices.

Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA.

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.

Molecular and cell biology of porcine HSP47 during wound healing: complete cDNA sequence and regulation of gene expression.

Heat shock protein (HSP) 47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. However, limited information is available regarding the regulation of HSP47 during wound healing. Using a polymerase chain reaction strategy, screening of a cDNA library, and RACE-polymerase chain reaction approaches, the sequence of a full-length porcine HSP47 cDNA has been identified. The cDNA contained 2096 bp that encodes for an 18 amino acid signal peptide and a mature protein coding region consisting of 401 amino acid residues. It also included 108 bp of the 5' noncoding region and a 731-bp 3' noncoding region. The deduced amino acid is 83% identical to chicken, 87% identical to mouse, 88% identical to rat, and 91% identical to human HSP47. It also shares between 26% and 30% identity with different members of the serine protease inhibitor superfamily. The protein contains a RDEL endoplasmic reticulum retention signal, and two potential glycosylation sites. All of these features are characteristic of HSP47 in higher vertebrates. Heat shock treatment of porcine fibroblasts led to up-regulation of HSP47 at both the transcriptional and translational levels. HSP47 protein levels were also up-regulated during skin wound healing in a pig model. Moreover, a higher molecular weight complex at approximately 140 Kda containing HSP47 was detected at the stage of healing that was coincident with the maximal transcriptional expression of HSP47 during wound healing in this animal model. Further investigation of how HSP47 is regulated during normal and abnormal skin wound healing may lead to new therapeutic approaches to improve the healing process.